Electrophysiological recording system and methods of using same

ABSTRACT

The disclosure provided herein provides systems, devices, and methods for analyzing cultured cells. Also disclosed herein are systems, devices, and methods for analyzing interactions between two different groups of cells. Also disclosed herein are systems and methods of producing and analyzing a three-dimensional cell culture.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a Divisional of U.S. application Ser. No.13/810,402, filed Mar. 28, 2013, which is a National Phase Applicationof International Application No. PCT/US2011/44128, filed Jul. 15, 2011,which claims priority to U.S. Provisional Application No. 61/399,694,filed on Jul. 16, 2010, which are hereby incorporated herein byreference in its entirety.

FIELD

The disclosed invention is generally related to analysis of culturedcells. More particularly, the disclosed invention is related toelectrophysiological analysis of cellular interactions between twogroups of cells, as well as the effects these cellular interactions haveon the electrophysiological characteristics of each respective group ofcells.

BACKGROUND

Measurement of extracellular signals from cells and tissues providesvital information in determining cellular excitability and conductivemechanisms. These signals are produced mostly by flow of ions likecalcium (Ca⁺), sodium (Na⁺), potassium (K⁺), and chloride (Cl⁻) throughcell membrane channels.

In vitro tissue biosynthesis is an invaluable resource not only fortissue-organ replacement, but also for studies of disease mechanisms aswell as for building high-throughput cell bioassay systems or biochipsfor pharmacological and proteomic studies. In vitro biosynthesis ofthree-dimensional (3D) multi-cellular structures that resemble adult ordifferentiated tissue has been tried for many years with limitedsuccess, mostly because organs enclose more than one cell type andbecause the existing amorphous bio-compatible materials do not permitpre-arrangement of different cell types with their appropriatefunctional architecture.

Multi-Electrode Array (MEA) devices have facilitated recordings ofextracellular electrical activity simultaneously from multiple sites, invitro and in vivo. Current MEA devices are designed for culturing andrecording from cells of one type or a mixture of different cell typescombined in a two dimensional scheme. While a major fraction of bodilytissues are composed of two or more cell types (e.g. glia and neurons inthe brain, fibroblasts, smooth muscle cells, endothelial cells andmyocytes in the heart and endothelial and smooth muscle cells in thearteries), there are no viable systems for studying thethree-dimensional interactions between such cells.

Electrical recordings in heart muscle can be made at surface cells usingoptical mapping or impaling glass microelectrodes. However, measurementsat deeper layers require the use of plunge electrodes, which can inducetissue damage.

There are similar challenges associated with physiological studies ofagonists, antagonists, and their corresponding receptors, such as inhigh-throughput drug screenings. Conventionally, such studies requirethe use of freshly isolated tissue samples, and results of the studiescan be adversely impacted by ischemia and other factors.

Thus, there is a need for systems of studying preparations where two ormore cell types can be co-cultured in a three-dimensional, controlledmanner. There is a further need for using such systems in improving theunderstanding of cell interaction mechanisms and to improve theartificial development of tissues. There is still a further need for anin vitro reconstituted system in which the architecture for multiplecell layers and types is produced with an electrical recording systemalready in place. There is still a further need for a cell-based assaythat permits performance of the high-throughput drug screenings andother physiological studies while avoiding the need for fresh tissuesamples and limiting the likelihood of events that adversely affect suchstudies.

SUMMARY

Disclosed herein are electrophysiological recording systems for analysisof cultured cells. The disclosed electrophysiological recording systemsinclude an electrophysiological recording device and a housing forsupporting the electrophysiological recording device. In one aspect, theelectrophysiological recording device has a porous membrane with top andbottom surfaces and a plurality of pores extending between the top andbottom surfaces. The porous membrane defines a first cell culture regiondisposed on the top surface and an opposed second cell culture regiondisposed on the bottom surface. In another aspect, theelectrophysiological recording device has a plurality of electrodespositioned thereon the porous membrane in between a first insulationlayer and a second insulation layer. The plurality of electrodes extendto the first cell culture region of the porous membrane. Recording endsof the electrodes measure electrical properties of cells cultured withinthe first and second cell culture regions, while the contact ends of theelectrodes are positioned in electrical communication with dataacquisition equipment.

In one aspect, the housing of the electrophysiological recording systemdefines a cell culture chamber. In this aspect, at least a portion ofthe porous membrane is mountable or otherwise configured for positioningwithin the cell culture chamber of the housing. In another aspect, thehousing of the electrophysiological recording system includes a firstbase support portion for supporting a recording portion of the porousmembrane, a second base support portion for supporting the contactportion of the porous membrane, and a first cover portion for attachmentover the first base portion. The first base portion and the first coverportion define respective openings that cooperate to define the cellculture chamber. The second base support portion supports the contactportion of the porous membrane such that at least a portion of the cellculture region of the porous membrane, including the recording portionof the porous membrane, overlies the opening of the first base supportportion. Optionally, the electrophysiological recording system includesmeans for rolling up the porous membrane to form a three-dimensionalcell culture.

Also disclosed are methods for analyzing cells using theelectrophysiological recording systems and devices described herein.

Also disclosed are methods of making tissue compositions using theelectrophysiological recording systems and devices described herein.

Also disclosed are methods of forming the electrophysiological recordingdevices described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the preferred embodiments of the inventionwill become more apparent in the detailed description in which referenceis made to the appended drawings wherein:

FIGS. 1A-1F depict an exploded view of an exemplary electrophysiologicalrecording system as described herein. FIG. 1A is an exploded view of theexemplary electrophysiological recording system. FIG. 1B is aperspective view of a recording device of the exemplaryelectrophysiological recording system of FIG. 1A. FIG. 1C is a partiallytransparent top view of the electrophysiological recording system ofFIG. 1A. FIG. 1D is a partially transparent side view of theelectrophysiological recording system of FIG. 1A. FIG. 1E is aperspective view of a gear assembly of the electrophysiologicalrecording system of FIG. 1A. FIG. 1F is a side view of a rod of a gearassembly and a porous membrane having a central aperture, as describedherein.

FIGS. 2A-2C depict another exemplary electrophysiological recordingsystem as described herein. FIG. 2A is a perspective view of theexemplary electrophysiological recording system. FIG. 2B is a partiallytransparent side view of the housing of the electrophysiologicalrecording system of FIG. 2A. FIG. 2C is a partially transparent top viewof the electrophysiological recording system of FIG. 2A.

FIGS. 3A-3E depict another exemplary electrophysiological recordingsystem as described herein. FIG. 3A is an exploded view of the exemplaryelectrophysiological recording system. FIG. 3B is a perspective view ofthe electrophysiological recording system of FIG. 3A, in an assembledconfiguration. FIG. 3C is a partially transparent side perspective viewof the electrophysiological recording system of FIG. 3A. FIG. 3D is apartially transparent top view of the electrophysiological recordingsystem of FIG. 3A. FIG. 3E is a partially transparent front view of theelectrophysiological recording system of FIG. 3A.

FIGS. 4A-4F depict an exemplary electrophysiological recording device asdescribed herein. FIG. 4A is a top view of the exemplaryelectrophysiological recording device. FIG. 4B is a close-up view of analignment hole of the electrophysiological recording device of FIG. 4A.FIG. 4C is a close-up view of an alignment marker of theelectrophysiological recording device of FIG. 4A. FIG. 4D is a close-upview of a cell culture region of the porous membrane of theelectrophysiological recording device of FIG. 4A. FIG. 4E is a close-upview of the recording ends of the electrodes of the electrophysiologicalrecording device of FIG. 4A. FIG. 4F is a close-up view of a stimulatingelectrode of the electrophysiological recording device of FIG. 4A.

FIG. 5 depicts a cross-sectional side view of the various layers of anexemplary electrophysiological recording device, as described herein.

FIGS. 6A-6L depict the various stages in the fabrication of an exemplaryelectrophysiological recording device, as described herein. FIG. 6A is aside perspective view of a glass slide. FIG. 6B is a side perspectiveview of a wafer formed by the application of a photoresist layer to theglass slide. FIG. 6C is a side perspective view of the wafer after aporous membrane has been applied to the photoresist layer. FIG. 6D is aside perspective view of the wafer after a first insulation layer hasbeen applied to the porous membrane. FIG. 6E is a side perspective viewof the wafer after the first insulation layer has been roughened throughan etching process. FIG. 6F is a side perspective view of the waferafter a layer of gold has been applied to the first insulation layer andthe porous membrane. FIG. 6G is a side perspective view of an exemplarymask pattern for forming a plurality of electrodes from the layer ofgold. FIG. 6H is a side perspective view of an exemplary mask patternfor forming an insulation layer of the electrophysiological recordingdevice. FIG. 6I is a side perspective view of the wafer after patterningof the plurality of electrodes. FIG. 6J is a side perspective view ofthe wafer after a second insulation layer is applied to the plurality ofelectrodes. FIG. 6K is a side perspective view of the wafer followingetching of the second insulation layer. FIG. 6L is a side perspectiveview of the electrophysiological recording device after separation fromthe glass slide and photoresist layer.

FIGS. 7A-7B provide images of the recording ends of exemplary electrodesas described herein. FIG. 7A is a microscopic image (200×magnification)of four exemplary electrodes within the recording portion of a porousmembrane as described herein. FIG. 7B is a microscopic image(2,000×magnification) of the recording end of one of the exemplaryelectrodes depicted in FIG. 7A.

FIG. 8 displays an exemplary process for forming a three-dimensionalcell culture, as described herein. In particular, FIG. 8 depicts the useof an exemplary gear assembly having a central rod for rolling up theporous membrane to form a three-dimensional cell culture of twodifferent groups of cells on opposite surfaces of a porous membrane thatcommunicate across the porous membrane, as described herein. FIG. 8further depicts the use of the rod to support the rolling up of theporous membrane and to permit recording of electrical properties of thetwo groups of cells following roll-up.

FIG. 9 displays a graph of the growth rate of cells cultured in Petridishes in which either a porous membrane as described herein or piecesof Sylgard were added to the culture medium, as compared to the growthrate of cells cultured alone in a Petri dish.

FIG. 10 describes an experiment that was used to confirm cell-to-cellcommunication through the porous membrane of an exemplaryelectrophysiological recording device as described herein. FIG. 10depicts the experimental setup and provides images demonstratingcommunication between cells located on opposite sides of the porousmembrane.

FIGS. 11A-11D display various recordings of cultured cardiomyocytesusing an exemplary electrophysiological recording device as describedherein. FIG. 11A displays recordings from an electrode array of theelectrophysiological recording device. FIG. 11B displays a singleelectrode recording at a constant beat rate. FIG. 11C displays aclose-up of an action potential measured by an electrode of theelectrode array. FIG. 11D displays a corresponding action potential asmeasured by a conventional multi-electrode array.

DETAILED DESCRIPTION OF THE INVENTION

The present invention can be understood more readily by reference to thefollowing detailed description, examples, drawings, and claims, andtheir previous and following description. However, before the presentdevices, systems, and/or methods are disclosed and described, it is tobe understood that this invention is not limited to the specificdevices, systems, and/or methods disclosed unless otherwise specified,and, as such, can, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularaspects only and is not intended to be limiting.

The following description of the invention is provided as an enablingteaching of the invention in its best, currently known embodiment. Tothis end, those skilled in the relevant art will recognize andappreciate that many changes can be made to the various aspects of theinvention described herein, while still obtaining the beneficial resultsof the present invention. It will also be apparent that some of thedesired benefits of the present invention can be obtained by selectingsome of the features of the present invention without utilizing otherfeatures. Accordingly, those who work in the art will recognize thatmany modifications and adaptations to the present invention are possibleand can even be desirable in certain circumstances and are a part of thepresent invention. Thus, the following description is provided asillustrative of the principles of the present invention and not inlimitation thereof

As used throughout, the singular forms “a,” “an” and “the” includeplural referents unless the context clearly dictates otherwise. Thus,for example, reference to “an electrode” can include two or more suchelectrodes unless the context indicates otherwise.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another aspect includes from the one particular value and/orto the other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another aspect. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint.

As used herein, the terms “optional” or “optionally” mean that thesubsequently described event or circumstance may or may not occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

As used herein, the term “or” as used herein means any one member of aparticular list and also includes any combination of members of thatlist.

Without the use of such exclusive terminology, the term “comprising” inthe claims shall allow for the inclusion of any additional elementirrespective of whether a given number of elements are enumerated in theclaim, or the addition of a feature could be regarded as transformingthe nature of an element set forth in the claims. Except as specificallydefined herein, all technical and scientific terms used herein are to begiven as broad a commonly understood meaning as possible whilemaintaining claim validity.

As used throughout, by “subject” is meant an individual. For example, a“subject” can be a mammal such as a primate, and, more preferably, ahuman. The term “subject” includes domesticated animals, such as cats,dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.),and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.). Forexample, the subject is an animal. In certain embodiments, the subjectis a human being. As used herein, a “subject” is the same as a“patient,” and the terms can be used interchangeably.

Described herein is an electrophysiological recording system foranalysis of cultured cells. With reference to FIGS. 1A-6L, theelectrophysiological recording system 100 comprises anelectrophysiological recording device 200 and a housing 300 forsupporting the electrophysiological recording device. Methods ofproducing and using the electrophysiological recording system 100 arealso described.

The Electrophysiological Recording Device

In one aspect, as shown in FIGS. 1A-5, the electrophysiologicalrecording device 200 can comprise a porous membrane 210 having a topsurface 212 and an opposed bottom surface 214. In this aspect, theporous membrane 210 can define a first cell culture region disposed on aselect portion of the top surface 212 and a second cell culture regiondisposed on a select portion of the bottom surface 214. It iscontemplated that the first cell culture region can overlie and have acorresponding size and shape to the second cell culture region. Thus,the first and second cell culture regions are together labeled in theFigures as a single element 216, even though it is understood that therespective cell culture regions are located on opposite surfaces of theporous membrane 210. In another aspect, the porous membrane 210 canfurther define a plurality of pores 230 extending between the opposedfirst and second cell culture regions on the respective opposed top andbottom surfaces 212, 214.

It is contemplated that the top surface 212 of the porous membrane 210can be configured to receive a first group of cells and that the bottomsurface 214 of the porous membrane 210 can be configured to receive asecond group of cells. Thus, in exemplary aspects, it is contemplatedthat the electrophysiological recording system 100 can comprise meansfor culturing cells on the top surface 212 and the bottom surface 214 ofthe porous membrane 210 within the first and second cell culture regions216. In these aspects, it is contemplated that the means for culturingcells on the top surface 212 and the bottom surface 214 of the porousmembrane 210 can comprise any known means for applying and culturingcells on a selected surface. It is further contemplated that the top andbottom surfaces 212, 214 of the porous membrane 210 can be selectivelycoated with one or more physiological substrates, such as, for exampleand without limitation, collagen, laminin, and the like, to increasecellular adhesion without compromising intercellular communication.

In a further aspect, the porous membrane 210 can comprise polycarbonate.In an exemplary aspect, the porous membrane 210 can be a polycarbonatetrack etch (PCTE) manufactured by STERLITECH CORPORATION. In anotherexemplary aspect, the porous membrane 210 can comprise a biodegradablematerial. In a further exemplary aspect the porous membrane 210 cancomprise a bioabsorbable material.

In an additional aspect, the porous membrane 210 can have a thicknessranging from about 1 micrometer to about 20 micrometers, more preferablyfrom about 5 micrometers to about 15 micrometers, and, most preferablyfrom about 8 micrometers to about 12 micrometers. In an exemplaryaspect, it is contemplated that the thickness of the porous membrane 210can be about 10 micrometers. In another aspect, each pore 230 of theplurality of pores of the porous membrane 210 can have a diameterranging from about 0.5 micrometers to about 15 micrometers, morepreferably from about 1 micrometer to about 10 micrometers, and mostpreferably from about 2 micrometers to about 5 micrometers. In anexemplary aspect, it is contemplated that each pore 230 of the pluralityof pores of the porous membrane 210 can have a diameter of about 3micrometers. In a further aspect, the plurality of pores 230 of theporous membrane 210 can be randomly scattered or otherwise presentwithin the opposed cell culture regions 216 at a density ranging fromabout 100,000 pores per square centimeter to about 300,000 pores persquare centimeter, more preferably from about 150,000 pores per squarecentimeter to about 250,000 pores per square centimeter, and mostpreferably from about 175,000 pores per square centimeter to about225,000 pores per square centimeter. In an exemplary aspect, theplurality of pores 230 of the porous membrane 210 can be randomlyscattered or otherwise present within the opposed cell culture regions216 at a density of about 200,000 pores per square centimeter.

It is contemplated that the plurality of pores 230 can be sized andpositioned such that cultured cells on the top surface 212 of the porousmembrane 210 can communicate with cultured cells on the opposed bottomsurface 214 of the porous membrane without the cultured cells on the topor bottom surface migrating across the porous membrane through theplurality of pores. It is further contemplated that the plurality ofpores 230 can permit formation of gap junctions between the culturedcells on the opposed top and bottom surfaces 212, 214 of the porousmembrane 210. It is further contemplated that the plurality of pores 230of the porous membrane 210 can be configured to permit communicationbetween cultured cells on the top surface 212 and bottom surface 214 ofthe porous membrane for three or more weeks. It is still furthercontemplated that, upon submersion of the porous membrane 210 in a cellculture medium within a conventional culture and/or recording chamber,the plurality of pores 230 can prevent the occurrence of ischemia incultured cells on the top or bottom surface 212, 214 of the porousmembrane. It is still further contemplated that the ability of thedisclosed electrophysiological recording system 100 to maintainintercellular communication between the layers of the porous membrane210, whether direct (gap junctions) or indirect (paracrine), can improveand promote maintenance of tissue homeostasis during cell culturing.

In an additional aspect, the electrophysiological recording device 200can comprise a first insulation layer 240. In this aspect, the firstinsulation layer 240 can be positioned thereon at least a portion of theporous membrane 210. In one aspect, the first insulation layer 240 canbe applied thereon the top surface 212 of the porous membrane 210 andtherein at least a portion of the pores 230 defined by the porousmembrane. In this aspect, as shown in FIG. 5, it is contemplated thatthe first insulation layer 240 can be applied as a continuous layeracross at least a portion of the top surface 212 of the porous membrane210 and within and across at least a portion of the pores 230 defined bythe porous membrane. Thus, it is contemplated that the first insulationlayer can extend from the top surface of the porous membrane into a porealong walls of the pore and across a bottom portion of the poresubstantially at the interface between the pore and the second surfaceof the porous membrane. In an exemplary aspect, the first insulationlayer 240 can be applied across substantially the entire top surface ofthe porous membrane. In this aspect, it is contemplated that the firstinsulation layer 240 can be applied thereon or within at least a portionof each pore of the plurality of pores 230 defined by the porousmembrane 210.

In a further aspect, the first insulation layer 240 can have a thicknessranging from about 100 nm to about 1 μm, more preferably from about 200nm to about 800 nm, and most preferably from about 300 nm to about 700nm. In an exemplary aspect, the first insulation layer 240 can have athickness of about 500 nm. Optionally, in an exemplary aspect, the firstinsulation layer 240 can comprise parylene, including, for example andwithout limitation, Parylene C, Parylene AF-4, Parylene SF, and ParyleneHT.

In another aspect, and with reference to FIGS. 4A-4F, theelectrophysiological recording device 200 can comprise an electrodearray comprising a plurality of electrodes 250. In this aspect, eachelectrode 250 of the plurality of electrodes can have a distal recordingend 252, a lead portion 254, and a proximal contact end 256. In anadditional aspect, the plurality of electrodes 250 can be positionedthereon the porous membrane 210 and the first insulation layer 240 suchthat each electrode of the plurality of electrodes extends to the firstcell culture region 216 of the porous membrane.

It is contemplated that the recording ends 252 of the plurality ofelectrodes 250 can be configured for measurement of electricalproperties of cells cultured within the first and second cell cultureregions 216 on the respective opposed top and bottom surfaces 212, 214of the porous membrane 210. It is further contemplated that therecording ends 252 of the plurality of electrodes 250 can be configuredto measure extracellular currents produced by action potentials of anyexcitable cell, such as, for example and without limitation, musclecells and neurons. It is still further contemplated that, throughappropriate distribution of the plurality of electrodes, the recordingends 252 of the plurality of electrodes 250 can be configured to measuretissue impedance, which can be indicative of the tightness and/ordensity of a particular tissue. It is still further contemplated that anion-sensitive electrode having ionic-exchange-sensitive resins can beincorporated into the electrophysiological recording system 100 topermit measurement of voltage changes, which can be indicative ofextracellular pH or other ionic concentrations.

It is further contemplated that the contact ends 256 of the plurality ofelectrodes 250 can be configured for electrical communication with dataacquisition equipment 400 using any known electrical communicationmeans. In an exemplary aspect, the contact ends 256 of the plurality ofelectrodes 250 can be configured for attachment to a one-pieceelectrical connector 410, such as, for example and without limitation, aSAMTEC SEI one-piece interface manufactured by SAMTEC, Inc. In thisaspect, it is contemplated that the one-piece electrical connector 410can be connected to additional data acquisition equipment 400 usingknown electrical communication means, including, for example and withoutlimitation, ribbon cables and the like. It is further contemplated thatthe one-piece electrical connector 410 can have a plurality of pins,such as, for example and without limitation, 25 pins, that areconfigured for electrical connection to respective contact ends 256 ofthe plurality of electrodes 250. In exemplary aspects, the contact ends256 of the plurality of electrodes 250 can be secured thereto a contactportion 218 of the porous membrane.

In another aspect, it is contemplated that portions of the first andsecond cell culture regions 216 proximate the recording ends 252 of theplurality of electrodes 250 can define a recording portion 220 of the ofthe porous membrane 210. It is contemplated that the recording portion220 can comprise a substantially circular region having a diameterranging from about 1 mm to about 15 mm, more preferably from about 2 mmto about 10 mm, and most preferably from about 4 mm to about 6 mm. In anexemplary aspect, the recording portion 220 can comprise a substantiallycircular region having a diameter of about 5 mm. In an exemplary aspect,a central portion of the recording portion 220 of the porous membrane210 can correspond to an area where no electrodes are positioned

In a further aspect, as shown in FIG. 4B, the contact portion 218 of theporous membrane 210 can comprise one or more alignment holes 222. Inthis aspect, it is contemplated that the one or more alignment holes 222of the contact portion 218 can be configured for alignment with one ormore corresponding holes 412 defined therein the one-piece electricalconnector 410, thereby permitting connection between the porous membrane210 and the one-piece electrical connector 410 by insertion of aconventional pin or other fastener through each alignment hole of thecontact portion and its corresponding hole in the electrical connector.In still a further aspect, as shown in FIG. 4C, the contact portion 218of the porous membrane 210 can comprise one or more alignment markers224. In this aspect, it is contemplated that the one or more alignmentmarkers 224 can correspond to guide markings that confirm properalignment of the porous membrane 210 during application of theelectrodes and various layers of the electrophysiological recordingdevice 200 as described herein.

In still a further aspect, the electrophysiological recording device 200can comprise a supporting film proximate one or more edges of the porousmembrane 210. It is contemplated that the supporting film can stabilizeand protect the porous membrane 210 during handling while also providingfurther insulation to the contact ends 256 of the plurality ofelectrodes 250. In an exemplary aspect, the supporting film can beKAPTON™ tape or film manufactured by DUPONT.

In one aspect, the lead portion 254 of each electrode 250 of theplurality of electrodes can have a width ranging from about 10 μm toabout 100 μm, more preferably from about 25 μm to about 75 μm, and mostpreferably from about 40 μm to about 60 μm. In an exemplary aspect, thelead portion 254 of each electrode 250 of the plurality of electrodescan have a width of about 50 μm. It is contemplated that, because thepores 230 of the porous membrane 210 can be randomly spaced within therecording portion 220, the pores in some areas of the recording portioncan be closer to one another than in other areas of the recordingportion. It is further contemplated that, in the areas of the recordingportion 220 with a greater concentration of pores 230, the electricalresistance will be higher than in areas of lesser pore concentration. Itis still further contemplated that the width of the recording ends 252and the lead portions 254 of the plurality of electrodes 250 can beselected to avoid such high resistance areas of the recording portion220 while still providing sufficient conduction of electrical signalsgenerated within the recording portion.

In another aspect, the recording end 252 of each electrode 250 of theplurality of electrodes can have a width ranging from about 50 μm toabout 200 μm, more preferably from about 75 μm to about 150 μm, and mostpreferably from about 90 μm to about 110 μm. In an exemplary aspect, therecording end 252 of each electrode 250 of the plurality of electrodescan have a width of about 100 μm. In one aspect, the respective contactends 256 of adjacent electrodes of the plurality of electrodes can bespaced apart from one another at at least one predetermined distance,and the respective recording ends of adjacent electrodes of theplurality of electrodes are spaced apart from one another at at leastone predetermined distance. In this aspect, within the recording portion220, it is contemplated that the respective recording ends 252 of theplurality of electrodes 250 can be spaced apart from recording ends ofadjacent electrodes by a distance ranging from between about 100 μm toabout 1 mm, more preferably from about 250 μm to about 750 μm, and mostpreferably from about 400 μm to about 600 μm. In an exemplary aspect,within the recording portion 220, the respective recording ends 252 ofthe plurality of electrodes 250 can be spaced apart from the recordingends of adjacent electrodes by about 500 μm. It is further contemplatedthat the contact ends 256 of the plurality of electrodes 250 can bespaced apart from one another within the contact portion 218 of theporous membrane 210 by at least 500 μm. In a further aspect, the contactends 256 of the plurality of electrodes 250 can have a width rangingfrom about 250 μm to about 750 μm, more preferably from about 350 μm toabout 650 μm, and most preferably from about 400 μm to about 600 μm.

In an exemplary aspect, the contact ends 256 of the plurality ofelectrodes 250 can have a width of about 500 μm. In yet another aspect,it is contemplated that the lengths of the contact ends 256 of theplurality of electrodes 250 can be varied as necessary for connectionthereto the data acquisition equipment 400. In this aspect, it iscontemplated that the lengths of the contact ends 256 of the pluralityof electrodes 250 can be greater than about 2 mm. It is furthercontemplated that the lengths of the contact ends 256 of the pluralityof electrodes 250 can be greater than about 3 mm.

In a further aspect, it is contemplated that each electrode 250 of theplurality of electrodes can have any cross-sectional profile, including,for example and without limitation, a square, rectangular, circular, orelliptical profile. However, it is further contemplated that electrodes250 having a square or rectangular profile can be more quickly andefficiently produced than electrodes having a circular or ellipticalprofile.

In an additional aspect, the electrophysiological recording system 100can comprise means for inducing an action potential in cells culturedwithin at least one of the first cell culture region and the second cellculture region. In an exemplary aspect, and with reference to FIG. 4F,the means for inducing an action potential can comprise at least onestimulating electrode 250 a among the plurality of electrodes 250 thatis configured to stimulate electrical activity within the recordingportion 220 of the porous membrane 210. In this aspect, the recordingend 252 a of the at least one stimulating electrode 250 a can have awidth of 100 μm to about 600 μm, more preferably from about 200 μm toabout 400 μm, and most preferably from about 250 μm to about 350 μm, andthe lead portion 254 a of the at least one stimulating electrode canhave a width ranging from about 50 μm to about 300 μm, more preferablyfrom about 100 μm to about 200 μm, and most preferably from about 125 μmto about 175 μm. In an exemplary aspect, the recording end 252 a of theat least one stimulating electrode 250 a can have a width of about 300μm, and the lead portion 254 a of the at least one stimulating electrodecan have a width of about 150 μm. In another exemplary aspect, it iscontemplated that the plurality of electrodes 250 can comprise 23electrodes, with three of the electrodes being stimulating electrodes.In a further aspect, it is contemplated that the plurality of electrodes250, as well as the at least one stimulating electrode, can be arrangedsymmetrically within the recording portion 220 of the porous membrane210.

In another exemplary aspect, the means for inducing an action potentialcan comprise one or more field electrodes. In this aspect, the one ormore field electrodes can be plaques of wires of any suitable conductivematerial, such as, for example and without limitation, platinum, thatare immersed in a cell culture solution close to the cultured cells. Itis contemplated that the means for inducing an action potential canfurther comprise an external amplifier for providing a current ofappropriate magnitude through the one or more field electrodes totrigger an action potential. The distance between the one or more fieldelectrodes can be selectively varied depending on the particular celltype to be stimulated. In an exemplary aspect, it is contemplated thatone or more field electrodes can be used to induce an action potentialin cultured cells using the method described in V. Sharma and L Tung,“Spatial Heterogeneity of transmembrane potential responses of singleguine-pig cardiac cells during electric field stimulation,” Journal ofPhysiology (2002), 542.2, pp 477-492, the disclosure of which is herebyincorporated by reference herein in its entirety.

In a further exemplary aspect, the means for inducing an actionpotential can comprise a light source. In this aspect, the light source,such as, for example and without limitation, a ultraviolet (UV) lightsource, can trigger action potentials in the cultured cells. It iscontemplated that the cultured cells can comprise cells that aretransfected with ChannelRhodopsin (such as ChannelRhodopsin-1,ChannelRhodpsin-2, or Volvox ChannelRhodopsin) or another compositionwith light-gated ion channels. For example, in one application, Helacells expressing Cx43 and transfected with ChannelRhodopsin-2 can becultured on an opposite surface of the porous membrane from myocytes.HeLa cells can form junctions with the myocytes, and a flash of UV lightcan open the light-gated ion channels of the ChannelRhodopsin, which inturn trigger action potentials in the myocytes. An exemplary method ofinducing an action potential in cells using ChannelRhodopsin-2 isdescribed in Boyden, E S, Zhan, F., Bamberg, E. nagel, G. andDeisseroth, K. Nat Neurosci. 8, 1263-1268 (2005), the disclosure ofwhich is hereby incorporated by reference herein in its entirety.

In still another aspect, the plurality of electrodes 250 can comprisegold. In this aspect, it is contemplated that the plurality ofelectrodes 250 can comprise gold and one or more elements for promotingadhesion of the electrodes to the first insulation layer 240 and/or theporous membrane 210. In an exemplary aspect, the plurality of electrodes250 can comprise gold, titanium oxide, and titanium, with the gold beingapplied over titanium oxide that has been applied over titanium. It iscontemplated that the high conductivity, chemical inertness, andmalleability of gold make it a desirable material for forming theplurality of electrodes 250. However, it is contemplated that any knownmaterial for forming electrodes can be used within the scope of thedisclosed electrophysiological recording device.

In a further aspect, the electrophysiological recording device 200 cancomprise a second insulation layer 270. In this aspect, the secondinsulation layer 270 can be positioned thereon each electrode 250 of theplurality of electrodes such that at least a portion of each electrodeis positioned therebetween the first insulation layer 240 and the secondinsulation layer 270. It is contemplated that the second insulationlayer 270 can be positioned thereon each electrode 250 of the pluralityof electrodes such that only the lead portion 254 of each electrode ispositioned therebetween the first insulation layer 240 and the secondinsulation layer. In other words, it is contemplated that the secondinsulation layer 270 can be applied such that the lead portion 254 ofeach electrode is insulated from the top and bottom and the recordingend 252 and contact end 256 of each electrode are only insulated fromthe bottom.

In one aspect, the second insulation layer 270 can be applied thereonthe plurality of electrodes 250 and a portion of the first insulationlayer 240. As shown in FIGS. 6K-6L, it is contemplated that the secondinsulation layer can comprise a plurality of spaced insulation elements272 that are configured to substantially completely overlie theplurality of electrodes 230. In another aspect, each insulation element272 of the plurality of spaced insulation elements of the secondinsulation layer 270 can have a width ranging between about 25 μm toabout 125 μm, more preferably from about 40 μm to about 100 μm, and mostpreferably from about 60 μm to about 80 μm. In an exemplary aspect, eachinsulation element 272 of the plurality of spaced insulation elements ofthe second insulation layer 270 can have a width of about 70 μm. It iscontemplated that the widths of the plurality of insulation elements 272of the second insulation layer 270 can be greater than the widths of theplurality of electrodes 250 to ensure complete insulation of theelectrodes.

In a further aspect, the second insulation layer 270 can have athickness ranging from about 100 nm to about 1 μm, more preferably fromabout 200 nm to about 800 nm, and most preferably from about 300 nm toabout 700 nm. In an exemplary aspect, the second insulation layer 270can have a thickness of about 500 nm. Optionally, in an exemplaryaspect, the second insulation layer 270 can comprise parylene,including, for example and without limitation, Parylene C, ParyleneAF-4, Parylene SF, and Parylene HT.

Methods of Producing the Disclosed Electrophysiological Recording Device

With reference to FIGS. 6A-6L, methods of producing theelectrophysiological recording device 200 are also disclosed. In oneaspect, a method of producing the electrophysiological recording devicecan comprise securing the porous membrane to a rigid substrate 600. Inthis aspect, as depicted in FIG. 6A, it is contemplated that the rigidsubstrate 600 can be, for example and without limitation, a conventionalglass or plastic slide. It is further contemplated that the porousmembrane can be cut to an appropriate size for overlying the rigidsubstrate 600.

In an exemplary aspect, as shown in FIGS. 6B-6C, the step of securingthe porous membrane to the rigid substrate can comprise applying aphotoresist layer 700 to the rigid substrate and then securing theporous membrane to the photoresist layer 700 such that the porousmembrane is coupled to the rigid substrate 600. In this aspect, it iscontemplated that the photoresist layer can have an appropriateviscosity for attaching the porous membrane to the rigid substrate whilealso blocking the pores of the porous membrane from the bottom. In oneaspect, the photoresist layer can comprise, for example and withoutlimitation, AZ 4620 photoresist. In an exemplary aspect, the photoresistlayer can be spun on the rigid substrate at about 2,500 rpm for about 50seconds. In another exemplary aspect, it is contemplated that thephotoresist layer can have a thickness ranging from about 8 to about 10μm. After the photoresist layer is applied to the rigid substrate, it iscontemplated that the resulting wafer can be baked at about 90° C. forabout five minutes on a hot plate to provide the photoresist layer witha smooth and uniform surface. It is contemplated that binding of theporous membrane to a rigid substrate can form a wafer that is ideal forlithography and configured to block the pores of the porous membranesuch that only one surface of the porous membrane can be coated.

In a further exemplary aspect, after the porous membrane is applied tothe photoresist layer, the resulting wafer (with the porous membrane)can be baked, thereby stretching the porous membrane and generatingbubble-like wrinkles on the porous membrane. In this aspect, thewrinkles on the porous membrane can be wiped off proximate the edges ofthe porous membrane. In an additional aspect, after the wrinkles on theporous membrane are removed, the wafer can be cooled.

In another aspect, and with reference to FIG. 6D, the method ofproducing the electrophysiological recording device can comprise coatingthe top surface of the porous membrane with the first insulation layerthrough a low pressure vapor deposition process (LPCVD). In this aspect,the porous membrane can be coated on all exposed surfaces, including thewalls of the pores. It is contemplated that the coating of the topsurface of the porous membrane using this isotopic process can block thepores of the porous membrane from the bottom surface of the porousmembrane, thereby insulating any additional layers that are deposited onthe first insulation layer from the bottom surface of the porousmembrane.

In a further aspect, as shown in FIG. 6E, the method of producing theelectrophysiological recording device can comprise dry etching the firstinsulation layer to roughen a top surface of the first insulation layer,thereby improving adhesion of layers applied on top of the firstinsulation layer. In an exemplary aspect, it is contemplated that thefirst insulation layer can be dry etched for about 30 s using oxygen asthe etching gas.

In another aspect, the method of producing the electrophysiologicalrecording device can comprise depositing one or more elements forpromoting adhesion of the electrodes to the first insulation layer. Inthis aspect, it is contemplated that a multi-cathode sputtering systemcan be used to deposit the one ore more elements for promoting adhesionof electrodes to the first insulation layer and then the plurality ofelectrodes. In an exemplary aspect, titanium (Ti) and titanium dioxide(TiO2) can be the one or more elements for promoting adhesion ofelectrodes, and the plurality of electrodes can comprise gold.

In still another aspect, the method of producing theelectrophysiological recording device can comprise preparing one or morepatterns for the plurality of electrodes and the first and/or secondinsulation layers. It is contemplated that the patterns of theelectrodes and insulation layers can be designed using known software,such as, for example and without limitation, L-Edit CAD software. Thepattern designs for the electrodes and insulator layers can be printedout on a glass plate, such as, for example and without limitation, a 5inch (12.7 cm) square glass plate, to thereby form one or more masks.Exemplary pattern designs for the electrodes and insulation layers arerespectively shown in FIGS. 6G-6H. Each mask can be oven-baked, cooled,and then placed in a glass container. In an exemplary aspect, each maskcan be oven-baked at 115° C. for about 20 minutes, then cooled for about2-3 minutes, and then placed in a glass container. In a further aspect,a photoresist developer can be applied over the one or more masks for apredetermined amount of time. In an exemplary aspect, AZ 300 MIF (AZElectronic Materials) can be poured slowly to cover the one or moremasks and left for about 10 seconds, at which point the one or moremasks can be removed and washed in DI water. In another exemplaryaspect, the one or more masks can be dipped in Cr 14-S chromium etch orother suitable composition until the mask becomes transparent, leavingbehind the pattern on the glass plate. The one or more masks can then bewashed in DI water and dried.

In still another aspect, and with reference to FIG. 6I, the method ofproducing the electrophysiological recording device can comprisepatterning the plurality of electrodes using lithographic techniques. Inthis aspect, the wafer can be spin coated with photoresist, such as, forexample and without limitation, Shipli 1813. In an exemplary aspect, thephotoresist can be applied at about 3,000 rpm for about 10 seconds andthen baked at about 90° C. for about 6 minutes. In an additional aspect,the photoresist layer can be exposed to Ultra Violet (UV) light throughthe mask. In this aspect, the wafer can be washed in developer 352solution or other suitable solution until the exposed photoresistdissolves. The wafer with the remaining photoresist pattern can bewashed in DI water, dried, post baked and again exposed to UV light. Ina further aspect, the wafer can then be washed in KI/I2 solution toremove the uncovered electrodes. The wafer can then be washed in DIwater, dipped in Buffered Oxide Etch (BOE) solution, and again washed inDI water. It is contemplated that the BOE solution can be used toselectively etch the titanium and titanium dioxide layers as describedherein. After the electrodes are patterned, the wafer can be washed indeveloper 352 or other suitable solution to remove the remainingpatterned photoresist.

As shown in FIG. 6J, in still another aspect, the method of producingthe electrophysiological recording device can comprise applying thesecond insulation layer thereon the plurality of electrodes and thefirst insulation layer as described herein.

In yet another aspect, the first and second insulation layers can bothbe patterned using lithographic techniques. The techniques used topattern the insulation layers are substantially the same as those usedto pattern the plurality of electrodes, as described herein. However, itis contemplated that the patterning of the insulation layers can be doneusing a different mask. It is further contemplated that the patterningof the second insulation layer can require an additional step ofaligning the second insulation layer with the existing pattern definedby the electrodes thereon the wafer. In one aspect, after an insulationlayer is properly aligned, the photoresist layer can be exposed to UVlight, washed in developer 352 or other suitable solution to dissolvethe exposed photoresist, rinsed in DI water, and then dried. In afurther aspect, the wafer can then be etched through an anisotropic,reactive ion etching (RIE) process to remove the uncovered portions ofthe insulation layers, leaving behind the final patterns of theinsulation layers. In an exemplary aspect, the wafer can be etchedthrough an RIE process in a Plasmalab 80 system for about 7 minutes toremove the uncovered portions of the insulation layers. FIG. 6K showsthe wafer following etching of the second insulation layer.

In another aspect, the method of producing the electrophysiologicalrecording device can comprise soaking the wafer in AZ400K solution orother suitable solution with gentle stirring to peel off the newlyformed electrophysiological recording device from the rigid substrate,as depicted in FIG. 6L. In this aspect, it is contemplated that, afterthe electrophysiological recording device was removed, it can be washedin DI water and then dried. In a further aspect, prior to cell culture,the electrophysiological recording device can be washed with PBS andethanol and then autoclaved so as to achieve sterilization.

The Housing

Also disclosed is a housing 300 defining a cell culture chamber 340. Inexemplary aspects, it is contemplated that at least a portion of therecording portion 220 of the porous membrane 210 can be mountable withinthe cell culture chamber 340 of the housing 300. In one aspect, and withreference to FIGS. 1A-3E, the housing 300 can comprise a first basesupport portion 310 and a second base support portion 320. In thisaspect, it is contemplated that the first base support portion 310 canbe configured to support the first and second cell culture regions 316of the porous membrane 210, while the second base support portion 320can be configured to support the contact portion 318 of the porousmembrane. It is further contemplated that the first and second basesupport portions 310, 320 can be shaped to permit the porous membrane210 to lie substantially flat on the first base support portion 310 tothereby permit cell culturing on either of the opposed first and secondcell culture regions 216 of the porous membrane.

In another aspect, the first base support portion 310 can define anopening 312. In this aspect, it is contemplated that the second basesupport portion 320 can be configured to support the porous membrane 210such that at least a portion of the recording portion 220 of the porousmembrane overlies the opening 312 of the first base support portion 310.It is further contemplated that the first base support portion 310 cancomprise a depressed region 314 that is configured to receive at least aportion of the porous membrane 210, such as, for example and withoutlimitation, the opposed first and second cell culture regions 316 of theporous membrane. In an exemplary aspect, the first base support portion310 can comprise a depressed region 314 that is depressed by about 10 μmfrom a top surface of the first base support portion. In this aspect,the size and shape of the depressed region 314 can substantiallycorrespond to the size and shape of a select portion of the porousmembrane 210.

Optionally, in another aspect, the first base support portion 310 andthe second base support portion 320 can be of unitary construction.Alternatively, in still another aspect, as shown in FIGS. 2A-2C, thefirst base support portion 310 and the second base support portion 320can be separate pieces of the housing 300. In this aspect, it iscontemplated that the first base support portion 310 can be configuredfor receipt within a conventional cell culture environment, such as, forexample and without limitation, a Petri dish, while the second basesupport portion 320 can be positioned outside the cell cultureenvironment. It is further contemplated that the design of the disclosedhousing 300 can permit sterile culturing of cells on the porous membrane210 in close proximity to computers and other data acquisition equipment400.

In a further aspect, the first base support portion 310 can define atleast one channel 316 in communication with the opening 312. In thisaspect, it is contemplated that the at least one channel 316 can permitaccess to the opening 312, and thus, the porous membrane 210, from aside edge of the first base support portion 310. It is furthercontemplated that, during culturing, the at least one channel 316 canpermit movement and/or circulation of culture media. In still anotheraspect, the first base support portion 310 and the second base supportportion 320 can each comprise a biocompatible polymer. For example, in anon-limiting aspect, it is contemplated that the first base supportportion 310 and the second base support portion 320 can each comprisepolychlorotrifluoroethylene (PCTFE).

In an additional aspect, the housing can comprise a first cover portion330. In this aspect, the first cover portion 330 can define an opening332. In a further aspect, the first cover portion 330 can be configuredfor attachment thereto the first base support portion 310 such that theopening 332 of the first cover portion is substantially aligned with theopening 312 of the first base support portion 310. In this aspect, it iscontemplated that the openings 312, 332 of the first base supportportion 310 and the first cover portion 330 can cooperate to define thecell culture chamber 340 for culturing a first group of cells on the topsurface 212 of the porous membrane 210 and a second group of cells onthe bottom surface 214 of the porous membrane. It is furthercontemplated that, in use, the opening 332 of the first cover portion330 can permit access to the top surface 212 of the porous membrane 210from above the porous membrane, while the opening 312 of the first basesupport portion 310 can permit access to the bottom surface 214 of theporous membrane from below the porous membrane. It is still furthercontemplated that the opening 332 of the first cover portion 330 canpermit imaging equipment to access the porous membrane 210 for analysisof cellular interactions within the cell culture chamber 340.

Optionally, in one aspect, and as shown in FIG. 3A, the cell culturechamber 340 can be surrounded by a sealant layer 350 for preventingleakage of cells or culture media from the cell culture chamber. In thisaspect, it is contemplated that the sealant layer 350 can comprise anelastomer, such as, for example and without limitation, a fast-curingsilicone elastomer. For example, in a non-limiting aspect, the sealantlayer 350 can comprise SYLGARD™ silicone elastomer manufactured by DOWCORNING.

In another aspect, the first cover portion 330 can define at least onechannel 334 in communication with the opening 332. In this aspect, it iscontemplated that the at least one channel 334 can permit access to theopening 332, and thus, the porous membrane 210, from a side edge of thefirst cover portion 330. It is further contemplated that, duringculturing, the at least one channel 334 can permit movement and/orcirculation of culture media. In an additional aspect, the first coverportion 330 can be selectively removable from the housing 300. In afurther aspect, the first cover portion 330 can comprise a biocompatiblepolymer. For example, in a non-limiting aspect, it is contemplated thatthe first cover portion 330 can comprise polychlorotrifluoroethylene(PCTFE).

In still another aspect, as shown in FIGS. 1A-1F, the housing 300 canoptionally comprise an intermediate support plate 360. In this aspect,the intermediate support plate 360 can be configured for operativecoupling to the contact portion 218 of the porous membrane 210. It iscontemplated that the intermediate support plate 360 can be integrallyconnected with a one-piece electrical connector 410 as described hereinfor operative connection to the contact ends 256 of the plurality ofelectrodes 250 within the contact portion 218 of the porous membrane210. In an additional aspect, the intermediate support plate 360 can beconfigured for attachment thereto the second base support portion 320.

In an exemplary aspect, the intermediate support plate 360 canoptionally comprise a gasket 362 configured to surround the contact ends256 of the plurality of electrodes 250 and the one-piece electricalconnector 410 within the contact portion 218 of the porous membrane 210.In this aspect, it is contemplated that the gasket 362 can cooperatewith the housing 300 to protect the contact ends 256 of the plurality ofelectrodes 250 and the one-piece electrical connector 410 from moisturewithin the housing, thereby preventing short circuits and signalduplication. In one aspect, the intermediate support plate 360 cancomprise an elastomer, such as, for example and without limitation, afast-curing silicone elastomer. For example, in a non-limiting aspect,the intermediate support plate 360 can comprise SYLGARD™ siliconeelastomer manufactured by DOW CORNING. It is contemplated that, when thehousing 300 does not comprise an intermediate support plate 360, thegasket 362 and the electrical connector 410 can be arranged directlythereon the second base support portion of the housing.

In a further aspect, the housing 300 can further comprise a second coverportion 370. In this aspect, the second cover portion 370 can beconfigured to overlie the second base support portion 320 of thehousing.

In another aspect, it is contemplated that the first base supportportion 310 and the first cover portion 330, as well as the second basesupport portion 320 and the second cover portion 370, can each have oneor more corresponding alignment holes 372. In this aspect, it is furthercontemplated that, in an operative position, the corresponding alignmentholes 372 of the first base support portion 310 and the first coverportion 330 are substantially axially aligned, and the second basesupport portion 320 and the second cover portion 370 are substantiallyaxially aligned. In an exemplary aspect, the corresponding alignmentholes 372 of the first base support portion 310 and the first coverportion 330 can be configured to receive a fastener such that the firstbase support portion and the first cover portion are secured to oneanother, and the corresponding alignment holes of the second basesupport portion 320 and the second cover portion 370 can be configuredto receive a fastener such that the second base support portion and thesecond cover portion are secured to one another. When the housing 300comprises an intermediate support plate 360, it is still furthercontemplated that the intermediate support plate 360 can have alignmentholes 372 that are aligned with corresponding alignment holes of thesecond base support portion 320 and the second cover portion 370 whenthe housing is placed in the operative position. In an exemplary aspect,the alignment holes 372 of the intermediate support plate 360 can beconfigured to receive a fastener such that the second base supportportion 320, the intermediate support plate, and the second coverportion 370 of the housing 300 can be secured to one another. In anotherexemplary aspect, it is contemplated that the fasteners used to securethe first base support portion 310 to the first cover portion 330 and tosecure the second base support portion 320, the intermediate supportplate 360, and the second cover portion 370 of the housing 300 can bescrews, such as for example and without limitation, nylon screws.Optionally, in another aspect, the second cover portion 370 can beintegrally formed with the intermediate support plate 360. In otherexemplary aspects, it is contemplated that the first base supportportion 310 can be secured to the first cover portion 330 using anyknown method or technique. Similarly, it is contemplated that the secondbase support portion 320, the intermediate support plate 360, and thesecond cover portion 370 can be secured to one another using any knownmethod or technique.

In various aspects, and with reference to FIGS. 1A-1F and 8, theelectrophysiological recording system 100 can further comprise means forrolling up the porous membrane 210 to form a three-dimensional cellculture 210 a. In these aspects, it is contemplated that the means forrolling up the porous membrane 210 can be configured to preventwobbling, compression, and/or over-stretching of the porous membraneduring formation of the three-dimensional cell culture 210 a. In oneaspect, the means for rolling up the porous membrane 210 can beintegrated into the housing 300. Alternatively, it is contemplated thatthe means for rolling up the porous membrane 210 can be an isolatedcomponent of the electrophysiological recording system 100.

In exemplary aspects, it is contemplated that the three-dimensional cellculture 210 a can have a substantially helical cross-sectional shape, asshown in FIG. 8. However, it is contemplated that the three-dimensionalcell culture 210 a can have any desired three-dimensional shape. It isfurther contemplated that the three-dimensional cell culture 210 a canpermit cells cultured on the opposed top and bottom surfaces 212, 214 ofthe porous membrane 210 to communicate either through the porousmembrane or through direct contact among the various layers of thethree-dimensional cell culture.

In one aspect, the means for rolling up the porous membrane 210 cancomprise a gear assembly 380. In this aspect, the gear assembly 380 cancomprise a rod 384 configured for attachment to at least a portion ofthe recording portion 220 of the porous membrane 210. In another aspect,the gear assembly 380 can comprise at least one gear 382 operativelycoupled to the rod 384 and rotatable about a rotation axis RA. In thisaspect, it is contemplated that rotation of the at least one gear 382can result in a corresponding rotation of the rod 384. It iscontemplated that each gear 382 of the at least one gear can have adiameter of about 1 mm. In this aspect, it is contemplated that theporous membrane 210 can be glued or otherwise attached to the rod 384such that rotation of the rod causes a corresponding rotation (androll-up) of the porous membrane. In an additional aspect, the rod 384can comprise an electrode 386 configured for measurement of electricalproperties of the three-dimensional cell culture 210 a. In this aspect,it is contemplated that the electrode 386 of the rod 384 can be furtherconfigured for electrical communication with the data acquisitionequipment 400. It is further contemplated that the porous membrane 210can be analyzed sequentially in its two-dimensional structure by theplurality of electrodes 250 of the electrophysiological recording device200 and in its three-dimensional structure 210 a by the plurality ofelectrodes 250 and/or the electrode 386 of the rod 384. In an exemplaryaspect, the electrode 386 of the rod 384 can be an ion-selectiveelectrode, such as, for example and without limitation, an electrodethat is selective for potassium (K+) ions.

In another aspect, and as shown in FIGS. 1A-1F, when the means forrolling up the porous membrane 210 is integrated into the housing 300,it is contemplated that the at least one gear 382 of the gear assembly380 can comprise a first gear 382 a and a second gear 382 b. In thisaspect, the means for rolling up the porous membrane 210 can furthercomprise a first groove 318 a and a second groove 318 b defined withinat least the first base support portion 310. Optionally, the firstgroove 318 a and the second groove 318 b can extend into, and be furtherdefined by, the first cover portion 330. It is contemplated that thefirst groove 318 a can be configured for receipt of the first gear 382 aand that the second groove 318 b can be configured for receipt of thesecond gear 382 b. It is further contemplated that the first groove 318a can be spaced apart from the second groove 318 b along the commonrotation axis RA of the first and second gears 382 a, 382 b. In afurther aspect, the first groove 318 a and the second groove 318 b canbe positioned on opposed sides of the opening 312 of the first basesupport portion 310.

In still another aspect, the first groove 318 a and the second groove318 b can be inclined at a predetermined angle. In this aspect, it iscontemplated that the predetermined angle can range from about 1 degreeto about 10 degrees and more preferably, from about 2 to about 3degrees. It is further contemplated that the incline of the first andsecond grooves 318 a, 318 b, as well as the diameter of the at least onegear 382, can determine the space between the various layers of theporous membrane 210 within the three-dimensional cell culture 210 a. Inexemplary aspects, it is contemplated that the space between the variouslayers of the porous membrane 210 can range from about 1 to about 20 μm,and more preferably from about 5 to about 10 μm. In a further aspect, itis contemplated that the first groove 318 a and second groove 318 b candefine a surface configured for complementary engagement with the atleast one gear 382, such as, for example and without limitation, asurface shaped to conform to teeth of the at least one gear, as shown inFIG. 1E.

In still another aspect, the rod 384 of the gear assembly 380 cancomprise stainless steel. In this aspect, it is contemplated that therod 384 can be a stainless steel needle, such as a stainless steelneedle having a diameter ranging from about 50 to about 150 μm, and morepreferably from about 80 to about 120 μm. In a further aspect, theelectrode 386 of the rod 384 can be glued or otherwise secured along thelength of the rod, and one or more connectors can be secured to the rodfor communication with the data acquisition equipment 400. Optionally,in one aspect, the rod 384 can be insulated with parylene or anotherknown polymer for creating a moisture and dielectric barrier around therod. In another aspect, it is contemplated that a tip 388 of theelectrode 386 of the rod 384 can be located at a center portion of therod and coated with a suitable resin. For example, and withoutlimitation, it is contemplated that the resin used to coat the tip 388of the electrode 386 can be any known liquid ion-exchange resin that canbe cationic or anionic, depending on the particular ion to bequantified, inside a polymer-based substrate, including thosemanufactured by Fluka (Sigma). In an additional aspect, it iscontemplated that the porous membrane 210 can be glued or otherwiseattached to the rod 384 before cells are cultured on the porousmembrane. Optionally, in still another aspect, as shown in FIG. 1F, theporous membrane 210 can define a central aperture 228 that issubstantially aligned with the tip 388 of the electrode 386 of the rod384 such that electrolyte diffusion is promoted within thethree-dimensional cell culture 210 a. In another aspect, the centralaperture 228 can have a length in a direction substantiallyperpendicular to the rotation axis RA of the gear assembly 380. In thisaspect, the length of the central aperture 228 can range from about 150μm to about 250 μm and, more preferably, can be about 200 μm.

Optionally, in an additional aspect, the housing 300 can furthercomprise at least one air suction tube 390 for removing air bubbles fromthe cell culture chamber 340. In this aspect, it is contemplated thatthe at least one air suction tube 390 can comprise a first suction tube390 a positioned below the porous membrane 210 and a second suction tube390 b positioned above the porous membrane. It is further contemplatedthat the at least one air suction tube 390 can be received withincorresponding bores 392 defined therein the first cover portion 330and/or the first base support portion 310 of the housing 300 such thatthe at least one air suction tube is positioned proximate the porousmembrane 210.

In additional exemplary aspects, it is contemplated that the materialsof the housing 300 can be configured to withstand temperatures up to atleast 200° C. such that the various elements of the housing can beautoclaved between uses. It is further contemplated that the materialsof the housing 300 can be configured to withstand conventionallaboratory sterilization treatments with alcohol and other known agents.

In exemplary aspects, the electrophysiological recording system canfurther comprise a Petri dish 500. In these aspects, it is contemplatedthat the cell culture chamber 340 of the housing 300 can be configuredfor receipt therein the Petri dish 500. More particularly, in anexemplary aspect, it is contemplated that the first base support portion310 and the first cover portion 330 of the housing 300 can be shaped forreceipt therein the Petri dish 500. It is further contemplated that,when the Petri dish 500 contains cell culture media, the first basesupport portion 310, which supports the porous membrane 210, and thefirst cover portion 330 of the housing can be configured for receipttherein the Petri dish 500 such that the cell culture media contacts theopposed first and second cell culture regions 216 of the porousmembrane. It one aspect, the Petri dish 500 can be a conventional Petridish manufactured from glass or plastic, including, for example andwithout limitation, a conventional 60 mm-diameter Petri dish.

Data Acquisition Equipment

In a further aspect, the electrophysiological recording system 100 canfurther comprise data acquisition equipment 400. In this aspect, asdescribed herein, the data acquisition equipment 400 can be placed inoperative electrical communication with the contact ends 256 of theplurality of electrodes 250 and/or with the electrode 386 of the gearassembly 380 using any known electrical communication means. Asdescribed herein, the data acquisition equipment 400 can comprise aone-piece electrical connector 410 for connection to the contact ends256 of the plurality of electrodes 250. It is contemplated that the dataacquisition equipment 400 can further comprise additional connectors forconnection to the electrode 386 of the gear assembly 380.

In another aspect, the data acquisition equipment 400 can furthercomprise a signal amplification and recording subsystem, a temperaturecontrol subsystem, noise reduction subsystem, and a computer-userinterface. Generally, in this aspect, the signal amplification andrecording subsystem can be configured to amplify the signals recorded bythe plurality of electrodes 250 to permit detailed analysis of thesignal deflections, the temperature control subsystem can be configuredto maintain the temperature of the cell culture chamber 340, the noisereduction subsystem can be configured to insulate and minimize signalnoise from the environment surrounding the cell culture chamber, and thecomputer-user interface can be configured to collect and visualize theelectrical signals generated by the cells cultured on the porousmembrane 210. It is contemplated that the housing 300 of theelectrophysiological recording system 100 can comprise openings inappropriate locations to permit electrical connection between theelectrodes 250 of the electrophysiological recording system and the dataacquisition equipment 400. Thus, for example, it is contemplated that anopening within the second cover portion 370 (or, alternatively, definedby the second cover portion and the second base support portion 320) canpermit connection of conventional cables or wires between a single-pieceelectrical connector 410 (coupled to the contact ends 256 of theplurality of electrodes 250) and external data acquisition equipment400.

In an additional aspect, the signal amplification and recordingsubsystem can comprise a known data acquisition system, such as, forexample and without limitation, a MEA60 amplifier system. In a furtheraspect, the signal amplification and recording subsystem can beconfigured to have a gain control mechanism. In this aspect, it iscontemplated that the gain control mechanism can comprise means forautomatically adjusting gain values according to the measured signalmagnitude and noise. In still another aspect, the signal amplificationand recording subsystem can be configured for connection to the contactends 256 of the plurality of electrodes 250 through a single-pieceinterface connector 410 or other connection means. In yet anotheraspect, the signal amplification and recording subsystem can beconfigured for connection to the computer-user interface. In thisaspect, it is contemplated that the signal amplification and recordingsubsystem can be configured for connection to the computer-userinterface using a known matrix cable system (MCS). In an exemplaryaspect, the signal amplification and recording subsystem can comprise ageneral ground connected to the signal amplification and recordingsubsystem for electrically grounding the overall electrophysiologicalrecording system. In this aspect, it is contemplated that the generalground can be, for example and without limitation, a chloridized silverwire that is immersed in cell culture media.

In still another aspect, the temperature control subsystem can comprisean incubator, including for example and without limitation, aheating/cooling micro-incubator stage (HCMIS) incubator, such as, forexample and without limitation, a PTC-10 (Peltier Temperature Control)system. It is contemplated that the incubator of the temperature controlsubsystem can be configured to maintain the porous membrane 210 at atemperature ranging from about 37° C. to about 38° C. It is furthercontemplated that any known temperature control mechanism can be usedfor purposes of the disclosed electrophysiological recording system 100.In exemplary aspects, it is contemplated that the temperature controlsubsystem can be positioned within the housing 300 of the disclosedelectrophysiological recording system 100.

In yet another aspect, the noise reduction subsystem can comprise aFaraday cage for receiving the electrophysiological recording device andhousing. In an exemplary aspect, the Faraday cage can comprise a boxhaving inner walls coated with aluminum mesh. It is contemplated thatthe Faraday cage can be configured to increase shielding against straynoise from the outside environment. In an additional aspect, when theelectrophysiological recording system 100 comprises a Petri dish 500,the noise reduction subsystem can further comprise an aluminum foillayer for shielding the Petri dish, thereby minimizing electromagneticnoise in the recorded signal.

In a further aspect, the computer-user interface can comprise a computerhaving a processor and a memory and a user-input means. In this aspect,it is contemplated that the computer-user interface can comprise akeyboard or other means for inputting information to the computer. It isfurther contemplated that the processor of the computer can beconfigured to perform steps required for observation and storage of thesignals recorded from the electrodes 250 of the electrophysiologicalrecording device 200 and/or gear assembly 380. It is still furthercontemplated that the memory of the computer can store conventionalsoftware for instructing the processor to observe and store the recordedsignals.

In an exemplary aspect, the memory can store conventional dataacquisition software, such as, for example and without limitation, MCRack software (Multi Channel Systems).

Methods for Analyzing Cells

In use, the electrophysiological recording system described herein canbe incorporated into various methods for analyzing cells. In one aspect,a method for analyzing cells can comprise applying a first group ofcells thereon the top surface of the porous membrane. In this aspect, itis contemplated that the first group of cells can be applied thereon thefirst cell culture region. In another aspect, the method for analyzingcells can comprise applying a second group of cells thereon the bottomsurface of the porous membrane. In this aspect, it is contemplated thatthe second group of cells can be applied thereon the second cell cultureregion. Optionally, the method for analyzing cells can comprise anyknown methods for optimizing acceptance of cells by the porous membrane.For example, it is contemplated that the method for analyzing cells canfurther comprise the step of applying one or more physiologicalsubstrates, such as, for example and without limitation, collagen,laminin, and the like, to at least one of the top or bottom surfaces ofthe porous membrane prior to application of the first and second groupsof cells.

In exemplary aspects, the first group of cells can be different from thesecond group of cells. Thus, it is contemplated that the first group ofcells can be of a first cell type and the second group of cells can beof a second cell type. In one exemplary aspect, the first group of cellscan be cardiac myocytes and the second group of cells can bemyofibroblasts or fibroblasts. In this aspect, it is contemplated thatanalysis of these two groups of cells can be used to study cell-to-cellcommunication in injured cardiac tissue. In another exemplary aspect,the first and second groups of cells can comprise one or more ofneurons, oligodendrocytes, and astrocytes. In this aspect, it iscontemplated that analysis of these groups of cells can be used to studycommunication maps in heterotypical neuronal tissue, as well as thepharmacological relevance of non-neuronal cells to conduction. In anadditional exemplary aspect, the first group of cells can comprisesmooth muscle cells and the second group of cells can compriseendothelial cells. In this aspect, it is contemplated that analysis ofthese two groups of cells can be used to study intercellularcommunication based on different connexins expressed and signaltransduction that occurs between the cells as the endothelial cells arestimulated and the muscle cells respond. It is further contemplated thatthe disclosed method can be used to analyze the pharmacology oflipidimia, as well as arterial agonists and antagonists, by monitoringchanges in shape of smooth muscle cells resulting from contraction orrelaxation after application of agonists or antagonists to theendothelial cell layer.

In an additional aspect, the method for analyzing cells can comprisepositioning the porous membrane in a cell culture medium. In thisaspect, when the porous membrane is secured to a housing as describedherein, the step of positioning the porous membrane in a cell culturemedium can comprise positioning at least the first base support portionof the housing within a container holding a cell culture medium, suchas, for example and without limitation, a Petri dish. Alternatively,when the first base support portion and the second base support portionof the housing are of unitary construction as described herein, then thestep of positioning the porous membrane in a cell culture medium cancomprise positioning at least the first base support portion and thesecond base support portion of the housing within a container holding acell culture medium, such as, for example and without limitation, aPetri dish.

In a further aspect, the method for analyzing cells can comprisedetecting at least one electrical signal at at least one recording endof the plurality of electrodes of the electrophysiological recordingdevice indicative of one or more electrical properties of the first andsecond groups of cells. In this aspect, it is contemplated that, whenthe porous membrane is connected to a plurality of electrodes and firstand second insulation layers to form an electrophysiological recordingdevice as described herein, the step of detecting at least oneelectrical signal can comprise positioning the porous membrane in thecell culture medium such that the recording ends of at least a portionof the electrodes contact the cell culture medium. It is furthercontemplated that, when the porous membrane is secured within a housingas described herein, the step of detecting at least one electricalsignal can comprise positioning the porous membrane such that therecording ends of at least a portion of the electrodes are positionedwithin the cell culture chamber defined by the first base supportportion and the first cover portion of the housing.

In still a further aspect, the method for analyzing cells can comprisetransmitting the at least one electrical signal to the data acquisitionequipment described herein. In this aspect, it is contemplated that thestep of transmitting the at least one electrical signal can compriseelectrically connecting the plurality of electrodes of theelectrophysiological recording device to data acquisition equipment asdescribed herein. More particularly, it is contemplated that the step ofdetecting at least one electrical signal can comprise electricallyconnecting the contact ends of the plurality of electrodes to the dataacquisition equipment. For example, and without limitation, the step ofdetecting at least one electrical signal can comprise electricallyconnecting an electrical connector as described herein to the contactends of the plurality of electrodes.

In another aspect, the method for analyzing cells can comprise receivingthe at least one electrical signal. In this aspect, it is contemplatedthat the data acquisition equipment described herein can be configuredto receive the at least one electrical signal. In an exemplary aspect,the step of receiving the at least one electrical signal can comprisestoring the at least one electrical signal in a memory as describedherein. In another exemplary aspect, the step of receiving the at leastone electrical signal can comprise processing the at least oneelectrical signal using software as described herein.

In an additional aspect, the method for analyzing cells can optionallycomprise the step of rolling up the porous membrane to form athree-dimensional cell culture. In this aspect, it is contemplated thatthe above steps of detecting, transmitting, and receiving the at leastone electrical signal can be performed while the porous membrane is in aflat configuration and then substantially repeated following formationof the three-dimensional cell culture. Alternatively, the steps ofdetecting, transmitting, and receiving the at least one electricalsignal can be performed only after the three-dimensional cell culture isformed as described herein. Thus, it is contemplated that the disclosedsystems and methods can permit controlled culturing of two groups ofcells prior to formation of the three-dimensional cell culture. In oneaspect, the step of rolling up the porous membrane can compriseattaching at least a portion of the recording portion of the porousmembrane to the rod of a gear assembly as described herein. In anotheraspect, the step of rolling up the porous membrane can comprise rotatingthe at least one gear of the gear assembly along a rotation axis suchthat the rod is rotated a corresponding amount, thereby rolling up theporous membrane. It is contemplated that, when the means for rolling upthe porous membrane comprises first and second grooves defined withinthe first base support portion as described herein, the step of rollingup the porous membrane can comprise rotating a first gear along thefirst groove and a second gear along the second groove. It is furthercontemplated that the respective first and second gears can be advancedalong the respective first and second grooves as the gears are rotated.For example, in an exemplary aspect, the respective first and secondgears can be advanced along the respective first and second groovestoward the second base support portion of the housing as the gears arerotated. It is still further contemplated that, when the porous membraneis secured within a housing as described herein, the step of rolling upthe porous membrane can comprise removing the first cover portion of thehousing prior to rotation of the at least one gear. In exemplaryaspects, the rotation of the at least one gear can be done manuallyuntil a desired degree of rotation and roll-up of the porous membrane isachieved.

As set forth herein, in various aspects, after formation of thethree-dimensional cell culture, the method of analyzing cells canfurther comprise detecting at least one electrical signal indicative ofone or more electrical properties of the first and second groups ofcells. In these aspects, it is contemplated that, when the porousmembrane is connected to a rod comprising an electrode as describedherein, the step of detecting at least one electrical signal cancomprise positioning the three-dimensional cell culture in the cellculture medium such that the electrode of the rod contacts the cellculture medium. It is further contemplated that the plurality ofelectrodes of the electrophysiological recording device can continue todetect the at least one electrical signal following formation of thethree-dimensional cell culture. It is further contemplated that, afterthe three-dimensional cell culture is positioned within the cell culturemedium, the method for analyzing cells can comprise transmitting the atleast one electrical signal to the data acquisition equipment describedherein. In one aspect, it is contemplated that the step of transmittingthe at least one electrical signal can comprise electrically connectingthe electrode of the rod to data acquisition equipment as describedherein. It is still further contemplated that, after thethree-dimensional cell culture is positioned within the cell culturemedium, the data acquisition equipment can receive the at least oneelectrical signal in the same manner described herein with respect tothe flat configuration of the porous membrane. Thus, it is contemplatedthat, after the three-dimensional cell culture is positioned within thecell culture medium, the at least one electrical signal can be stored ina memory as described herein and/or can be processed using software asdescribed herein.

In exemplary aspects, it is contemplated that, following culture of thefirst and second groups of cells as described herein, theelectrophysiological recording device, including the porous membrane towhich the first and second groups of cells are attached, can beselectively removable from the housing for use in other applications.

In exemplary aspects, it is contemplated that the above-describedmethods of analyzing cells can be used to evaluate particular tissuecompositions for various applications. In one exemplary aspect, themethods can be used to evaluate compositions comprising normal and/orhypertrophied cardiac cells. In another exemplary aspect, the methodscan be used to evaluate compositions comprising cells obtained from anischaemic/injured brain cortex. In an additional exemplary aspect, themethods can be used to evaluate compositions comprising normal and/orhypertrophied arterial tissues. In a further exemplary aspect, themethods can be used to evaluate compositions comprising injuredendothelial tissue cells, such as cells obtained from an injured portionof the digestive tract. In still another exemplary aspect, the methodscan be used to evaluate compositions comprising pulmonary alveoli. Inthis aspect, it is contemplated that the tissue composition can beevaluated for formation of independent capillary beds.

In additional exemplary aspects, it is contemplated that theabove-described methods of analyzing cells can be used to create acell-based assay that permits performance of a high-throughput drugscreening. In these aspects, it is contemplated that the cell-basedassay can comprise a plurality of substantially identical cell culturechambers as described herein can be set in line. Each cell culturechamber can contain substantially identical cellular combinations ofcells, such as, for example and without limitation, endothelial andsmooth muscle cells. Electrical recordings from each respective cellculture chamber can be used to evaluate changes in input resistance thatoccur in each cell culture chamber. Each cell culture chamber can beconnected to various drug concentrations through known microcirculationsystems and methods. Thus, it is contemplated that the disclosed systemsand methods can provide time synchrony and homogeneity in analyzingparticular drug concentrations in various cell cultures. Additionally,it is contemplated that in the disclosed methods, intercellularcommunication between the different cell types can be restricted byreducing the number of pores and not changing a cell genome duringconventional siRNA or transfection procedures.

In a further exemplary aspect, it is contemplated that bioelectricalimpedance measurements of selected groups of cells can be obtained usingthe disclosed systems and methods. In this aspect, it is contemplatedthat the plurality of electrodes of the electrophysiological recordingsystem can comprise a large electrode (reference electrode) surroundedby a plurality of smaller electrodes. It is contemplated thatbioelectrical impedance measurements can be based on calculating thefrequency-dependent electrical impedance of cell-covered electrodes,such as, for example and without limitation, gold electrodes asdescribed herein, along the time course of an experiment to therebymonitor cellular morphological changes in cultured cells. It iscontemplated that cultured cells on top of the electrodes can behave asinsulating particles that hinder unrestricted current flow from theelectrode into cell culture media, thereby increasing the overallelectrode impedance. It is contemplated that at certain currentfrequencies, an applied current can couple through the porous membranecapacitance and cross the cellular layer on trans-cellular pathways. Formost frequencies, it is contemplated that this current will bypass thecellular bodies. Along these para-cellular pathways, it is contemplatedthat the current will travel first through thin tunnels between formedbetween the porous membrane and the plurality of electrodes. Theelectrical resistance between cells (Rb) and the resistance betweencells and the porous membrane (α) can be monitored over time using theabove-described system. Through monitoring of these properties, it iscontemplated that the contraction properties of various tissuecompositions can be measured in vitro. It is contemplated that suchcontractions can induce cell flattening, thereby contributing to adecrease in Rb and an increase in α. It is further contemplated that thepores of the membrane can reduce the effective current that theplurality of electrodes detect. It is contemplated that the porousmembrane can be selectively modified to obtain an optimal or desiredsurface for applying a recording/reference electrode. An exemplarytechnique for such any analysis is described in Arndt S, Seebach J,Psathaki K, Galla H-J, Wegener J. Bioelectrical impedance assay tomonitor changes in cell shape during apoptosis. Biosensors andBioelectronics 2004;19:583-94, which disclosure is incorporated hereinby reference in its entirety.

The invention will be further described with reference to the followingexamples; however, it is to be understood that the invention is notlimited to such examples. Rather, in view of the present disclosure thatdescribes the current best mode for practicing the invention, manymodifications and variations would present themselves to those of skillin the art without departing from the scope and spirit of thisinvention. All changes, modifications, and variations coming within themeaning and range of equivalency of the claims are to be consideredwithin their scope.

EXAMPLES

Experiment 1

Mouse myocytes were cultured on a first surface of a porous membrane.Tumor cells (HeLa) were cultured on a second surface of the porousmembrane. Prior to application of the myocytes to the first surface andthe tumor cells to the second surface, both surfaces were coated with amixture of collagen, laminin and fibronectin. The cultured myocytes beatspontaneously for a week. Recordings from these cells using a disclosedelectrophysiological recording system are presented in FIGS. 11A-11D.Lucifer yellow injected in cells at one side of the membrane diffusedthrough gap junctions into cells on the other side, demonstratingfunctional interaction between the tumor cells and the cardiac myocytes.

Experiment 2

50,000±2500 HeLa WT cells were plated in three sets of five 35 mm Petridishes each. The first set was used as control. Small 33 mm pieces ofthe disclosed porous membrane and of Sylgard were placed in the secondand third set, respectively. Cell number was calculated everyday fromeach set using a hemocytometer during the following 5 consecutive days.

FIG. 9 shows a time vs. cell concentration plot for the three sets. Allthree curves increase exponentially until days 4-5, when the cellconcentration rate reduces or even drops in one case (with Sylgard). Thegrowth rate for the Sylgard and the porous membrane were comparable andwere both larger than the control growth rate.

Experiment 3

On day 0, HeLa Cx43 cells were stained with DiI fluorescent dye andplated on the first surface of a porous membrane as described herein. Onday 1, the membrane was turned over, and the second surface of theporous membrane was plated with another set of HeLa cells. These cellswere loaded with CellTracker™ Green CMFDA (also known as Green Tracker).Following application of the two cellular layers, the porous membranepreparation was incubated for 6-8 hours. After incubation, thepreparation was observed under the microscope in UV light through redand green filters, with red-stained cells with green dye indicatingcoupling across the two cellular layers through the porous membrane.

The HeLa Cx43 cells were successfully plated on both sides of the porousmembrane and showed normal cell growth on visual inspection under themicroscope. Each cell layer was viewed after modifying the microscopefocus. Using the red filter, the microscope was focused to thered-stained cells' level. The filter was changed to green and cells thatappeared focused with the green dye were spotted. The microscope wasthen focused to the top layer to identify the prospective donor cellacross the membrane. Multiple pairs of cells that transferred the greendye across the membrane were spotted. FIGS. 11A-11D show one such case.

Experiment 4

An electrophysiological recording device was positioned within a housingas disclosed herein. The complete and assembled system was washed withPBS and ethanol, and dry-autoclaved at 120° C. The system was thenplaced in a 60 mm Petri dish to maintain the cells in culture medium.Before plating the cells, the porous membrane was hydrated and treatedwith fibronectin. Hydration of the porous membrane was achieved byleaving the system in PBS solution for 6-8 hours. Fluid soaked throughthe air filled pores of the porous membrane, allowing fluid movementacross the membrane. The porous membrane was soaked for 1 hour in 2μg/ml fibronectin solution to promote cell adhesion to the membrane.Neonatal myocytes were isolated and the equivalent to 6-8 hearts wasplated on the first surface of the porous membrane. 300 μMBromodeoxyuridine (BrdU) was added to the cell culture media (DMEM) torestrict fibroblast growth. (DMEM solution: HyQ DMEM/High Glucose; 10%FBS Fetal Bovine Serum, 1% Pen Strep [10000 ui Penicilin, 10,000 ug/mlStreptomicin, 25 ug/ml Amphoterecin B], 1% L-Glutamine 200 mM and 1%Non-essential-amino acids).

After the cells were plated, the housing was left in the incubator (37°C., 6% CO2, moisture) for 36-48 hours. During this time, the cellculture media was changed every 48 hours.

Before electrophysiological recordings, the culture media was exchangedfor OptiMEM (DMEM with Hepes pH buffer) and incubated for 60-90 minutesfor acclimatization. After acclimatization, the 60 mm Petri dishcontaining the housing was positioned in a customized HCMISmicro-incubator. The micro-incubator maintained the preparation attemperatures between 37-38° C. A single-piece electrical connectorprovided an electrical connection between twenty electrodes of theelecrophysiological recording device and a multi-electrode arrayamplifier. The signals from the preparation were recorded and storedusing MCRack software on a computer. Recordings were taken at a samplingrate of 5 kHz.

After recordings were completed, the system was unplugged from the dataacquisition equipment, the cell culture media in the Petri dish waschanged back to 300 μM BrdU DMEM solution and left for incubation.

FIGS. 11A-11D show the signals recorded from the 20 recording electrodeson the electrophysiological recording device on day 1 after plating.Signals recorded at electrodes 21, 24, 33, 34, 44 and 51 can be clearlyidentified as action potentials (refer to FIG. 11A). Action potentialshape was comparable to signals recorded from cardiomyocyte preparationsin a standard MEA. FIGS. 11C-11D show one such selected pair of actionpotentials from the porous membrane and a standard MEA. FIG. 11B shows aconstant beat rate of 33 beats per minute. The identified actionpotentials measured a signal to noise ratio (SNR) of 8.6 and peak topeak voltage (Vpp) of 200 μV.

Experiment 5

An electrophysiological recording device as described herein wasfabricated. The electrophysiological recording device was developed ontop of a porous membrane made from PCTE. The fabrication steps are asshown in FIGS. 6A-6L.

Initially, a glass slide with a clean surface was selected. AZ P4620photoresist was spun on the glass slide at 2500 rpm for 50 seconds toform an 8-10 um thick photoresist layer. The resulting wafer was thenbaked at 90° C. for 5 minutes on a hot plate to give the photoresist asmooth and uniform surface. The porous membrane was cut appropriatelyand placed on the photoresist layer.

The wafer and porous membrane were baked together, generatingbubble-like wrinkles on the membrane. These wrinkles were wiped offgently, and the wafer and porous membrane were cooled. The porousmembrane was then coated with parylene using a SCS PDS 2010 parylenecoater. The low pressure vapor deposition process (LPCVD) consumed 0.88grams of parylene dimer to produce a uniform parylene coating (layer 1)of 445 nm thickness on all exposed surfaces including the walls of thepores.

The parylene layer was etched for 30 seconds with an Oxford Plasmalab 80Plus system using oxygen as the etching gas to improve adhesion of thegold electrodes. A TMV SS-40C-IV, Multi Cathode Sputtering system wasused to deposit Titanium oxide (TiO) followed by gold (Au) over theparylene surface. The deposited titanium oxide and gold layer had atotal thickness of 93.1 nm. Lithography was used to pattern themetal/conducting layer.

The wafer was spin coated with photoresist (Shipli 1813) at 3000 rpm for10 seconds and baked at 90° C. for 6 minutes (not shown). The electrodepattern was designed using L-Edit CAD software. The pattern design wasprinted on a 4″ square glass plate in the

Electromask MM250 pattern generator. The photoresist layer was thenexposed to Ultra Violet (UV) light for 8 seconds through the mask. Themask blocked the UV light, exposing only the non-shaded regions of themask. The wafer was then washed in developer 352 solution for 50 secondsuntil the exposed photoresist dissolved. The wafer with the remainingphotoresist pattern was washed in deionized water (DI water), dried andexposed to UV light for another 8 seconds. Since gold was shielding thephotoresist at the bottom layer, only the patterned photoresist layerwas exposed. The wafer was then washed in (Potassium Iodide/Iodine)KI/I2 solution for about 30 seconds to remove the uncovered gold. Thewafer was then washed in DI water, dipped in Buffered Oxide Etch (BOE)solution for 10 seconds and again washed in DI water. Once theelectrodes were patterned, the solution was washed in developer 352 for50 seconds to remove the remaining patterned photoresist.

To insulate the lead portions of the electrodes, another layer ofparylene (layer 2) was deposited and patterned. Deposition was done inthe SCS PDS 2010 parylene coater using 0.92 grams of dimer to obtain asecond coat of thickness 650 nm. Patterning of both parylene layers wasdone by lithography as above, but with a different mask and anadditional alignment step with the existing gold pattern on the wafer.Mask-wafer alignment was done using a EVG 420 mask aligner. Oncealigned, the photoresist layer was exposed to UV light for 8 seconds,washed in developer 352 to dissolve the exposed photoresist, followed byrinsing in DI water and drying. The wafer was then etched, implementingan anisotropic process—Reactive Ion Etching (RIE)—in the OxfordPlasmalab 80. RIE was carried out for 7 minutes to remove the uncoveredparylene layers and pattern the parylene.

The wafer was then soaked in AZ400K solution with gently stirring topeel off the newly formed electrophysiological recording device. Afterthe electrophysiological recording device came off, it was washed in DIwater and dried. The electrophysiological recording device was handledusing forceps and stored in a 35 mm Petri dish. The electrophysiologicalrecording device was washed with phosphate buffered saline (PBS) andethanol, followed by an autoclaving process to sterilize it before cellculture.

Equivalents

Although several embodiments of the invention have been disclosed in theforegoing specification, it is understood by those skilled in the artthat many modifications and other embodiments of the invention will cometo mind to which the invention pertains, having the benefit of theteaching presented in the foregoing description and associated drawings.It is thus understood that the invention is not limited to the specificembodiments disclosed hereinabove, and that many modifications and otherembodiments are intended to be included within the scope of the appendedclaims. Moreover, although specific terms are employed herein, as wellas in the claims which follow, they are used only in a generic anddescriptive sense, and not for the purposes of limiting the describedinvention, nor the claims which follow.

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

What is claimed is:
 1. A method of forming a three-dimensional cellculture, comprising: positioning a porous membrane having a firstsurface and an opposed second surface in a substantially planarconfiguration, the porous membrane defining a first cell culture regiondisposed on a select portion of the first surface and a second cellculture region disposed on a select portion of the second surface, theporous membrane defining a plurality of pores extending between thefirst surface and the second surface within the opposed first and secondcell culture regions, wherein an electrode array is secured to theporous membrane, the electrode array comprising a plurality ofelectrodes having distal recording ends that are secured to the porousmembrane and extend to the first cell culture region of the porousmembrane, and wherein the porous membrane and the electrode arraycooperate to form a cell culture structure; applying a first group ofcells thereon the first surface of the porous membrane within the firstcell culture region; applying a second group of cells thereon theopposed second surface of the porous membrane within the second cellculture region; and rolling up the porous membrane and the electrodearray to form a three-dimensional cell culture structure having a spiralcross-sectional shape.
 2. The method of claim 1, further comprisingapplying one or more physiological substrates to at least one of thefirst surface or the second surface of the porous membrane prior toapplying the first group of cells and the second group of cells.
 3. Themethod of claim 1, wherein the first group of cells differs from thesecond group of cells, wherein the first group of cells comprises afirst cell type and the second group of cells comprises a second celltype different than the first cell type.
 4. The method of claim 3,wherein the first cell type comprises cardiac myocytes.
 5. The method ofclaim 4, wherein the second cell type comprises at least one ofmyofibroblasts or fibroblasts.
 6. The method of claim 1, wherein thefirst group of cells and the second group of cells comprise one or moreof neurons, oligodendrocytes, and astrocytes.
 7. The method of claim 1,wherein the first group of cells comprises smooth muscle cells.
 8. Themethod of claim 7, wherein the second group of cells comprisesendothelial cells.
 9. The method of claim 1, further comprisingpositioning the porous membrane in a cell culture chamber.
 10. Themethod of claim 9, further comprising securing the porous membrane to ahousing, wherein the housing comprises: a first base support portiondefining an opening; a second base support portion that supports theporous membrane such that at least a portion of the opposed first andsecond cell culture regions of the porous membrane overlie the openingof the first base support portion; and a first cover portion defining anopening, the first cover portion being attached to the first basesupport portion such that the opening of the first cover portion issubstantially aligned with the opening of the first base supportportion, wherein the respective openings of the first base supportportion and the first cover portion cooperate to define the cell culturechamber of the housing.
 11. The method of claim 10, further comprisingpositioning at least the first base support portion of the housingwithin a container holding the cell culture chamber.
 12. The method ofclaim 11, wherein the container is a Petri dish.
 13. The method of claim1, further comprising detecting at least one electrical signal at atleast one recording end of the plurality of electrodes of the electrodearray indicative of one or more electrical properties of the first andsecond regions of cells.
 14. The method of claim 13, further comprisingpositioning the porous membrane in a cell culture chamber such that theat least one recording end of the plurality of electrodes contacts thecell culture chamber.
 15. The method of claim 13, further comprisingtransmitting the at least one electrical signal to data acquisitionequipment.
 16. The method of claim 15, wherein the at least oneelectrical signal is transmitted to the data acquisition equipment priorto formation of the three-dimensional cell culture structure.
 17. Themethod of claim 15, wherein the at least one electrical signal istransmitted to the data acquisition equipment following formation of thethree-dimensional cell culture structure.
 18. The method of claim 1,further comprising inducing an action potential in cells cultured withinat least one of the first cell culture region and the second cellculture region.
 19. The method of claim 18, wherein the step of inducingan action potential comprises activating a light source configured totrigger the action potential.
 20. The method of claim 19, wherein the atleast one of the first group of cells and the second group of cellscomprises cells having light-gated ion channels.
 21. The method of claim20, wherein the cells having light-gated ion channels comprise cellsthat are transfected with ChannelRhodopsin.
 22. The method of claim 21,wherein the first group of cells comprises myocytes, and wherein thesecond group of cells comprises Hela cells expressing Cx43 andtransfected with ChannelRhodopsin-2.
 23. The method of claim 1, whereinportions of the first and second cell culture regions proximate therecording ends of the plurality of electrodes define a recording portionof the porous membrane.
 24. The method of claim 23, further comprisinginducing an action potential using at least one electrode of theplurality of electrodes, wherein the at least one electrode of theplurality of electrodes is configured to stimulate electrical activitywithin the recording portion of the porous membrane.
 25. The method ofclaim 1, wherein the porous membrane and the electrode array are rolledup using a gear assembly comprising: a rod attached to at least aportion of the recording portion of the porous membrane; and at leastone gear coupled to the rod and rotatable about a rotation axis, whereinrotation of the at least one gear about the rotation axis results in acorresponding rotation of the rod.
 26. The method of claim 25, furthercomprising attaching at least a portion of the recording portion of theporous membrane to the rod of the gear assembly.
 27. The method of claim26, further comprising rotating the at least one gear of the gearassembly relative to the rotation axis to effect rotation of the rod androlling up of the porous membrane.
 28. The method of claim 26, whereinthe rod comprises an electrode, wherein the porous membrane defines acentral aperture that is substantially aligned with a tip of theelectrode of the rod, and wherein the method further comprises using thecentral aperture to diffuse electrolytes within the three-dimensionalcell culture.
 29. A method of forming a three-dimensional cell culture,comprising: applying a first group of cells thereon a first surface of aporous membrane positioned in a substantially planar configuration, theporous membrane having a second surface opposed from the first surface,the porous membrane defining a first cell culture region disposed on aselect portion of the first surface and an opposed second cell cultureregion disposed on a select portion of the second surface, the porousmembrane further defining a plurality of pores extending between theopposed first and second cell culture regions on the respective opposedfirst and second surfaces, wherein the porous membrane is configured tocooperate with an electrode array to form an electrophysiologicalrecording device, the electrode array comprising a patterned layer thatdefines a plurality of electrodes, each electrode of the plurality ofelectrodes having a distal recording end and a proximal contact end,wherein the distal recording end of each electrode of the plurality ofelectrodes is secured to the porous membrane and extends to the firstcell culture region of the porous membrane, wherein the recording endsof the plurality of electrodes are configured for measurement ofelectrical properties of cells cultured within the first and second cellculture regions on the respective opposed first and second surfaces,wherein the contact ends of the plurality of electrodes are configuredfor electrical communication with data acquisition equipment; applying asecond group of cells thereon the opposed second surface of the porousmembrane; and rolling up the porous membrane and the electrode array ofthe electrophysiological recording device to form a three-dimensionalcell culture structure having a spiral cross-sectional shape.